Journal: Journal of Endocrinology

Article Title: Local adiponectin treatment reduces atherosclerotic plaque size in rabbits

doi: 10.1677/joe-06-0173

Figure Lengend Snippet: Figure 1 Serum adiponectin concentrations in rabbits. 1, Aorta non-injured rabbits on normal cholesterol diet; 2, aorta injured rabbits on high-cholesterol diet before adenovirus transfer; 3, after transfer Ad-bgal through intima; 4, after transfer Ad-APN through intima; 5, after transfer Ad-bgal through adventitia; 6, After transfer Ad-APN through adventitia. The symbol * indicates significant difference (P!0.01) vs serum adiponectin concentrations in aorta non-injured rabbits on normal cholesterol diet; † compared with rabbits before adenovirus transfer; and ‡ compared with rabbits after Ad-bgal transfer. The values are meansGS.E.M.

Article Snippet: Following blocking with 5% non-fat milk, the blots were incubated with anti-His tag mouse monoclonal antibody (1:1000 dilution, Applygen Technologies Inc., Beijing, China) and rabbit adiponectin polyclonal antibody (1:100 dilution, Boster, Wuhan, Hubei, China) at 4 8C overnight.

Techniques:

Journal: Journal of Endocrinology

Article Title: Local adiponectin treatment reduces atherosclerotic plaque size in rabbits

doi: 10.1677/joe-06-0173

Figure Lengend Snippet: Figure 2 SDS-PAGE (A) and western blot analysis of the supernatant of cultured Pichia pastoris (B, using anti-His tag monoclonal antibody; C, using adiponectin polyclonal antibody). Line 1 is a blank pGAPZa Pichia vector (not containing adiponectin gene) sample control and lines 2–4 are samples from the supernatant of cultured Pichia pastoris containing the recombinant rabbit adiponectin gene. Line 5 contained the molecular weight markers (not visible in image of blot). Both anti-His antibody and adiponectin polyclonal antibody detected a single predominant protein at z42 kDa (containing 9.3 kDa a-factor signal peptide and 2.5 kDa C-terminal His-tag).

Article Snippet: Following blocking with 5% non-fat milk, the blots were incubated with anti-His tag mouse monoclonal antibody (1:1000 dilution, Applygen Technologies Inc., Beijing, China) and rabbit adiponectin polyclonal antibody (1:100 dilution, Boster, Wuhan, Hubei, China) at 4 8C overnight.

Techniques: SDS Page, Western Blot, Cell Culture, Plasmid Preparation, Control, Recombinant, Molecular Weight

Journal: Journal of Endocrinology

Article Title: Local adiponectin treatment reduces atherosclerotic plaque size in rabbits

doi: 10.1677/joe-06-0173

Figure Lengend Snippet: Figure 5 Effect of Ad-APN treatment on adhesion molecules in atherosclerotic rabbits 14 days after recombinant adenovirus transfer. A, Immunohistochemical detection of adiponectin, VCAM-1 and ICAM-1 in atherosclerotic lesions. Representative images are shown. 1, Ad-bgal transfer through intima. 2, Ad-APN transfer through intima. 3, Ad-bgal transfer through adventitia. 4, Ad-APN transfer through adventitia. The magnification is 400. B, mRNA expression of VCAM-1 and ICAM-1 in abdominal aortic tissue in Ad-bgal or Ad-APN-treated rabbits. The values are meansGS.E.M.

Article Snippet: Following blocking with 5% non-fat milk, the blots were incubated with anti-His tag mouse monoclonal antibody (1:1000 dilution, Applygen Technologies Inc., Beijing, China) and rabbit adiponectin polyclonal antibody (1:100 dilution, Boster, Wuhan, Hubei, China) at 4 8C overnight.

Techniques: Recombinant, Immunohistochemical staining, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Prolonged Chronic Consumption of a High Fat with Sucrose Diet Alters the Morphology of the Small Intestine

doi: 10.3390/ijms22147280

Figure Lengend Snippet: Antibodies used with their sources and dilutions.

Article Snippet: Adiponectin , Boster Biological Technology, Pleasanton, CA, USA; code PA2014-1 , 1:50.

Techniques:

Journal: Arthritis Research & Therapy

Article Title: Potential therapeutic antibodies targeting specific adiponectin isoforms in rheumatoid arthritis

doi: 10.1186/s13075-018-1736-3

Figure Lengend Snippet: Detection of serum adiponectin isoforms with monoclonal antibodies (mAbs) against recombinant human adiponectin. a Western blot to screen mAbs detecting different adiponectin isoforms. Human serum was loaded into each lane of the gel and resolved by polyacrylamide gel electrophoresis (PAGE) and then transferred to a membrane. Each lane of the membrane was then separately cut and incubated with each mAb from hybridoma culture supernatant. After the secondary antibody was probed, the cut membrane was combined to form one sheet and developed with ECL solution. Eleven different mAbs from culture supernatant were tested to investigate their recognition patterns of adiponectin isoforms in serum. The mAb KH7–33, in lane 3, was selected as a representative recognizing only the middle molecular weight (MMW) isoform of adiponectin. The mAb KH7–41, in lane 4, was selected as a representative recognizing both the MMW and low molecular weight (LMW) isoforms of adiponectin. The mAb KH4–8, in lane 8, was selected as a representative mAb recognizing both the MMW and HMW isoforms of adiponectin. b Comparison of the mAb recognition patterns of adiponectin isoforms in human, mouse, and rat sera by Western blot. Human, mouse, and rat sera were separated by sodium dodecyl sulfate–PAGE. All three mAbs (KH7–33, KH7–41, and KH4–8) recognized the rat and mouse adiponectin MMW isoform. Lane 1, KH7–41; lane 2, KH7–33; lane 3, KH4–8; lane 4, commercial antibody against human adiponectin (Boster Immunoleader cat. no. PB9001) or mouse/rat adiponectin (Boster Immunoleader cat. no. PB9011). Mouse and rat serum were obtained via heart puncture of male BALB/c mice and Sprague Dawley rats (8 weeks old), respectively. Human serum was obtained from a male volunteer (55 years old)

Article Snippet: Lane 1, KH7–41; lane 2, KH7–33; lane 3, KH4–8; lane 4, commercial antibody against human adiponectin (Boster Immunoleader cat. no. PB9001) or mouse/rat adiponectin (Boster Immunoleader cat. no. PB9011).

Techniques: Bioprocessing, Recombinant, Western Blot, Polyacrylamide Gel Electrophoresis, Membrane, Incubation, Molecular Weight, Comparison

Journal: Arthritis Research & Therapy

Article Title: Potential therapeutic antibodies targeting specific adiponectin isoforms in rheumatoid arthritis

doi: 10.1186/s13075-018-1736-3

Figure Lengend Snippet: Immunohistological assays in human tissues with monoclonal antibodies (mAbs). To determine the pattern of recognition of adiponectin isoforms in human tissues by mAbs, a normal human adipose tissue was immunostained with mAbs (KH7–41, KH7–33, and KH4–8) (200×, scale bar = 25 μm). mAbs recognized adiponectin in the nucleus of adipocytes (blue arrow) and in vessels and endothelial cells (red arrow). b Human lung, kidney, and pancreas were stained with the KH7–41, KH7–33, and KH4–8 mAbs (100×). Abbreviations: HMW high molecular weight, LMW low molecular weight, MMW middle molecular weight

Article Snippet: Lane 1, KH7–41; lane 2, KH7–33; lane 3, KH4–8; lane 4, commercial antibody against human adiponectin (Boster Immunoleader cat. no. PB9001) or mouse/rat adiponectin (Boster Immunoleader cat. no. PB9011).

Techniques: Bioprocessing, Staining, High Molecular Weight, Molecular Weight

Journal: Arthritis Research & Therapy

Article Title: Potential therapeutic antibodies targeting specific adiponectin isoforms in rheumatoid arthritis

doi: 10.1186/s13075-018-1736-3

Figure Lengend Snippet: Inhibition of adiponectin-mediated gene expression in vitro by monoclonal antibodies (mAbs). To test the ability of the mAb KH4–8 to block adiponectin function, ( a ) human osteoblasts and ( b ) human umbilical vein endothelial cells (HUVECs) were treated with adiponectin (ADIPO) or KH4–8 mAb (mAb) or both. The mAb (~120 μg/mL) and recombinant adiponectin (2.5 μg/mL) were mixed and incubated for 1 h before being used to treat cells. After 24-h treatment, the culture supernatants were collected and frozen, and interleukin-6 (IL-6) and IL-8 were measured by using enzyme-linked immunosorbent assay (ELISA) (R&D Systems, Minneapolis, MN, USA). The experiments were performed in quadruplicate. The data shown are representative of three independent experiments, and similar results were obtained with all three mAbs. Values are expressed as mean ± standard error of the mean. The expression levels of the factors were compared between groups by using the Mann–Whitney test. * P <0.05, ** P <0.01 versus the untreated group, # P <0.05, ## P <0.01 versus the group treated with adiponectin and mAb

Article Snippet: Lane 1, KH7–41; lane 2, KH7–33; lane 3, KH4–8; lane 4, commercial antibody against human adiponectin (Boster Immunoleader cat. no. PB9001) or mouse/rat adiponectin (Boster Immunoleader cat. no. PB9011).

Techniques: Inhibition, Gene Expression, In Vitro, Bioprocessing, Blocking Assay, Recombinant, Incubation, Enzyme-linked Immunosorbent Assay, Expressing, MANN-WHITNEY

Journal: Arthritis Research & Therapy

Article Title: Potential therapeutic antibodies targeting specific adiponectin isoforms in rheumatoid arthritis

doi: 10.1186/s13075-018-1736-3

Figure Lengend Snippet: Epitope mapping of monoclonal antibody (mAb) KH4–8 against adiponectin. To identify the epitope-recognizing site of the KH4–8 mAb, PEPperMAP ® technology was performed as described in the Methods. Human adiponectin was translated into linear 15–amino acid peptides with a peptide-peptide overlap of 14 amino acids. Human adiponectin peptide microarrays were incubated with mouse mAb KH4–8 at different concentrations followed by staining with secondary goat anti-mouse IgG (H + L) DyLight680 antibody. The light intensity was read by a reader. The amino acid sequence QQNHYD (139–144) was confirmed from among the full 244–amino acid sequence to be the epitope of adiponectin

Article Snippet: Lane 1, KH7–41; lane 2, KH7–33; lane 3, KH4–8; lane 4, commercial antibody against human adiponectin (Boster Immunoleader cat. no. PB9001) or mouse/rat adiponectin (Boster Immunoleader cat. no. PB9011).

Techniques: Incubation, Staining, Sequencing

Journal: Arthritis Research & Therapy

Article Title: Potential therapeutic antibodies targeting specific adiponectin isoforms in rheumatoid arthritis

doi: 10.1186/s13075-018-1736-3

Figure Lengend Snippet: Anti-inflammatory effect of monoclonal antibodies (mAbs) KH4–8 and KH7–33 on the expression of serum pro-inflammatory cytokines of the collagen-induced arthritis mouse model. The serum levels of adiponectin, interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), and receptor activator of nuclear factor-kappa Β ligand (RANKL) were analyzed by using the Luminex system. Values are expressed as mean ± standard error of the mean. The expression levels of the factors were compared between groups ( n = 8) by using the Mann–Whitney test. ** P <0.01, * P <0.05 versus normal (NOR) group, and ## P <0.01, ## P <0.05 versus the control (CON) group. Abbreviations: ns not significant, pre prednisolone

Article Snippet: Lane 1, KH7–41; lane 2, KH7–33; lane 3, KH4–8; lane 4, commercial antibody against human adiponectin (Boster Immunoleader cat. no. PB9001) or mouse/rat adiponectin (Boster Immunoleader cat. no. PB9011).

Techniques: Bioprocessing, Expressing, Luminex, MANN-WHITNEY, Control

Journal: Arthritis Research & Therapy

Article Title: Potential therapeutic antibodies targeting specific adiponectin isoforms in rheumatoid arthritis

doi: 10.1186/s13075-018-1736-3

Figure Lengend Snippet: Anti-adiponectin antibodies reduce the histological signs of inflammation. The upper and lower panels present hematoxylin and eosin (H&E) staining and immunostaining against mouse adiponectin of mouse knee joints (n = 8), respectively. a Normal, b control, saline-treated arthritic, c KH7–33-treated arthritic, d KH4–8-treated arthritic, and ( e ) prednisolone-treated arthritic mice. Tissue structure was visualized by using H&E staining (original magnification, 40×). Scale bar = 2 mm. f Arthritic symptoms were evaluated by scoring the degree of inflammation on H&E histological sections of knee joints as described in the Methods. Small blue squares on H&E staining are magnified in the upper right corner (400×). Abbreviations: C cartilage, F femur, M meniscus, S subchondral bone, T tibia. In the lower panel, immunohistochemistry (IHC) reveals adiponectin expression in collagen-induced arthritis mouse joints (200×). The increased adiponectin expression level observed on IHC was not decreased by monoclonal antibody treatment. Adiponectin immunostaining score level was evaluated as described in the Methods. Results are presented as the mean of experiments (± standard error of the mean indicated by error bar) (one-way analysis of variance followed by Dunn’s multiple comparison test). *** P <0.001 versus the normal (NOR) group and # P <0.05, ## P <0.01 versus the control (CON) group. Abbreviation: pre prednisolone

Article Snippet: Lane 1, KH7–41; lane 2, KH7–33; lane 3, KH4–8; lane 4, commercial antibody against human adiponectin (Boster Immunoleader cat. no. PB9001) or mouse/rat adiponectin (Boster Immunoleader cat. no. PB9011).

Techniques: Staining, Immunostaining, Control, Saline, Immunohistochemistry, Expressing, Comparison

Journal: International Journal of Molecular Sciences

Article Title: Prolonged Chronic Consumption of a High Fat with Sucrose Diet Alters the Morphology of the Small Intestine

doi: 10.3390/ijms22147280

Figure Lengend Snippet: ( A , C ) Immunohistochemistry of adiponectin and adiponectin receptor of proximal tract of the small intestine. (Original magnification: 10×; scale bar: 100 μm). ( B , D ) The expression of adiponectin and its receptor was significantly reduced in HFD w/Suc mice compared to the SD group. * p < 0.05. These images are representative of n = 5 SD, n = 10 HFD w/Suc mice.

Article Snippet: Adiponectin , Boster Biological Technology, Pleasanton, CA, USA; code PA2014-1 , 1:50.

Techniques: Immunohistochemistry, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Prolonged Chronic Consumption of a High Fat with Sucrose Diet Alters the Morphology of the Small Intestine

doi: 10.3390/ijms22147280

Figure Lengend Snippet: Antibodies used with their sources and dilutions.

Article Snippet: Adiponectin , Boster Biological Technology, Pleasanton, CA, USA; code PA2014-1 , 1:50.

Techniques:

Journal: Arthritis Research & Therapy

Article Title: Potential therapeutic antibodies targeting specific adiponectin isoforms in rheumatoid arthritis

doi: 10.1186/s13075-018-1736-3

Figure Lengend Snippet: Detection of serum adiponectin isoforms with monoclonal antibodies (mAbs) against recombinant human adiponectin. a Western blot to screen mAbs detecting different adiponectin isoforms. Human serum was loaded into each lane of the gel and resolved by polyacrylamide gel electrophoresis (PAGE) and then transferred to a membrane. Each lane of the membrane was then separately cut and incubated with each mAb from hybridoma culture supernatant. After the secondary antibody was probed, the cut membrane was combined to form one sheet and developed with ECL solution. Eleven different mAbs from culture supernatant were tested to investigate their recognition patterns of adiponectin isoforms in serum. The mAb KH7–33, in lane 3, was selected as a representative recognizing only the middle molecular weight (MMW) isoform of adiponectin. The mAb KH7–41, in lane 4, was selected as a representative recognizing both the MMW and low molecular weight (LMW) isoforms of adiponectin. The mAb KH4–8, in lane 8, was selected as a representative mAb recognizing both the MMW and HMW isoforms of adiponectin. b Comparison of the mAb recognition patterns of adiponectin isoforms in human, mouse, and rat sera by Western blot. Human, mouse, and rat sera were separated by sodium dodecyl sulfate–PAGE. All three mAbs (KH7–33, KH7–41, and KH4–8) recognized the rat and mouse adiponectin MMW isoform. Lane 1, KH7–41; lane 2, KH7–33; lane 3, KH4–8; lane 4, commercial antibody against human adiponectin (Boster Immunoleader cat. no. PB9001) or mouse/rat adiponectin (Boster Immunoleader cat. no. PB9011). Mouse and rat serum were obtained via heart puncture of male BALB/c mice and Sprague Dawley rats (8 weeks old), respectively. Human serum was obtained from a male volunteer (55 years old)

Article Snippet: Lane 1, KH7–41; lane 2, KH7–33; lane 3, KH4–8; lane 4, commercial antibody against human adiponectin (Boster Immunoleader cat. no. PB9001) or mouse/rat adiponectin (Boster Immunoleader cat. no. PB9011).

Techniques: Bioprocessing, Recombinant, Western Blot, Polyacrylamide Gel Electrophoresis, Membrane, Incubation, Molecular Weight, Comparison

Journal: Arthritis Research & Therapy

Article Title: Potential therapeutic antibodies targeting specific adiponectin isoforms in rheumatoid arthritis

doi: 10.1186/s13075-018-1736-3

Figure Lengend Snippet: Immunohistological assays in human tissues with monoclonal antibodies (mAbs). To determine the pattern of recognition of adiponectin isoforms in human tissues by mAbs, a normal human adipose tissue was immunostained with mAbs (KH7–41, KH7–33, and KH4–8) (200×, scale bar = 25 μm). mAbs recognized adiponectin in the nucleus of adipocytes (blue arrow) and in vessels and endothelial cells (red arrow). b Human lung, kidney, and pancreas were stained with the KH7–41, KH7–33, and KH4–8 mAbs (100×). Abbreviations: HMW high molecular weight, LMW low molecular weight, MMW middle molecular weight

Article Snippet: Lane 1, KH7–41; lane 2, KH7–33; lane 3, KH4–8; lane 4, commercial antibody against human adiponectin (Boster Immunoleader cat. no. PB9001) or mouse/rat adiponectin (Boster Immunoleader cat. no. PB9011).

Techniques: Bioprocessing, Staining, High Molecular Weight, Molecular Weight

Journal: Arthritis Research & Therapy

Article Title: Potential therapeutic antibodies targeting specific adiponectin isoforms in rheumatoid arthritis

doi: 10.1186/s13075-018-1736-3

Figure Lengend Snippet: Inhibition of adiponectin-mediated gene expression in vitro by monoclonal antibodies (mAbs). To test the ability of the mAb KH4–8 to block adiponectin function, ( a ) human osteoblasts and ( b ) human umbilical vein endothelial cells (HUVECs) were treated with adiponectin (ADIPO) or KH4–8 mAb (mAb) or both. The mAb (~120 μg/mL) and recombinant adiponectin (2.5 μg/mL) were mixed and incubated for 1 h before being used to treat cells. After 24-h treatment, the culture supernatants were collected and frozen, and interleukin-6 (IL-6) and IL-8 were measured by using enzyme-linked immunosorbent assay (ELISA) (R&D Systems, Minneapolis, MN, USA). The experiments were performed in quadruplicate. The data shown are representative of three independent experiments, and similar results were obtained with all three mAbs. Values are expressed as mean ± standard error of the mean. The expression levels of the factors were compared between groups by using the Mann–Whitney test. * P <0.05, ** P <0.01 versus the untreated group, # P <0.05, ## P <0.01 versus the group treated with adiponectin and mAb

Article Snippet: Lane 1, KH7–41; lane 2, KH7–33; lane 3, KH4–8; lane 4, commercial antibody against human adiponectin (Boster Immunoleader cat. no. PB9001) or mouse/rat adiponectin (Boster Immunoleader cat. no. PB9011).

Techniques: Inhibition, Gene Expression, In Vitro, Bioprocessing, Blocking Assay, Recombinant, Incubation, Enzyme-linked Immunosorbent Assay, Expressing, MANN-WHITNEY

Journal: Arthritis Research & Therapy

Article Title: Potential therapeutic antibodies targeting specific adiponectin isoforms in rheumatoid arthritis

doi: 10.1186/s13075-018-1736-3

Figure Lengend Snippet: Epitope mapping of monoclonal antibody (mAb) KH4–8 against adiponectin. To identify the epitope-recognizing site of the KH4–8 mAb, PEPperMAP ® technology was performed as described in the Methods. Human adiponectin was translated into linear 15–amino acid peptides with a peptide-peptide overlap of 14 amino acids. Human adiponectin peptide microarrays were incubated with mouse mAb KH4–8 at different concentrations followed by staining with secondary goat anti-mouse IgG (H + L) DyLight680 antibody. The light intensity was read by a reader. The amino acid sequence QQNHYD (139–144) was confirmed from among the full 244–amino acid sequence to be the epitope of adiponectin

Article Snippet: Lane 1, KH7–41; lane 2, KH7–33; lane 3, KH4–8; lane 4, commercial antibody against human adiponectin (Boster Immunoleader cat. no. PB9001) or mouse/rat adiponectin (Boster Immunoleader cat. no. PB9011).

Techniques: Incubation, Staining, Sequencing

Journal: Arthritis Research & Therapy

Article Title: Potential therapeutic antibodies targeting specific adiponectin isoforms in rheumatoid arthritis

doi: 10.1186/s13075-018-1736-3

Figure Lengend Snippet: Anti-inflammatory effect of monoclonal antibodies (mAbs) KH4–8 and KH7–33 on the expression of serum pro-inflammatory cytokines of the collagen-induced arthritis mouse model. The serum levels of adiponectin, interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), and receptor activator of nuclear factor-kappa Β ligand (RANKL) were analyzed by using the Luminex system. Values are expressed as mean ± standard error of the mean. The expression levels of the factors were compared between groups ( n = 8) by using the Mann–Whitney test. ** P <0.01, * P <0.05 versus normal (NOR) group, and ## P <0.01, ## P <0.05 versus the control (CON) group. Abbreviations: ns not significant, pre prednisolone

Article Snippet: Lane 1, KH7–41; lane 2, KH7–33; lane 3, KH4–8; lane 4, commercial antibody against human adiponectin (Boster Immunoleader cat. no. PB9001) or mouse/rat adiponectin (Boster Immunoleader cat. no. PB9011).

Techniques: Bioprocessing, Expressing, Luminex, MANN-WHITNEY, Control

Journal: Arthritis Research & Therapy

Article Title: Potential therapeutic antibodies targeting specific adiponectin isoforms in rheumatoid arthritis

doi: 10.1186/s13075-018-1736-3

Figure Lengend Snippet: Anti-adiponectin antibodies reduce the histological signs of inflammation. The upper and lower panels present hematoxylin and eosin (H&E) staining and immunostaining against mouse adiponectin of mouse knee joints (n = 8), respectively. a Normal, b control, saline-treated arthritic, c KH7–33-treated arthritic, d KH4–8-treated arthritic, and ( e ) prednisolone-treated arthritic mice. Tissue structure was visualized by using H&E staining (original magnification, 40×). Scale bar = 2 mm. f Arthritic symptoms were evaluated by scoring the degree of inflammation on H&E histological sections of knee joints as described in the Methods. Small blue squares on H&E staining are magnified in the upper right corner (400×). Abbreviations: C cartilage, F femur, M meniscus, S subchondral bone, T tibia. In the lower panel, immunohistochemistry (IHC) reveals adiponectin expression in collagen-induced arthritis mouse joints (200×). The increased adiponectin expression level observed on IHC was not decreased by monoclonal antibody treatment. Adiponectin immunostaining score level was evaluated as described in the Methods. Results are presented as the mean of experiments (± standard error of the mean indicated by error bar) (one-way analysis of variance followed by Dunn’s multiple comparison test). *** P <0.001 versus the normal (NOR) group and # P <0.05, ## P <0.01 versus the control (CON) group. Abbreviation: pre prednisolone

Article Snippet: Lane 1, KH7–41; lane 2, KH7–33; lane 3, KH4–8; lane 4, commercial antibody against human adiponectin (Boster Immunoleader cat. no. PB9001) or mouse/rat adiponectin (Boster Immunoleader cat. no. PB9011).

Techniques: Staining, Immunostaining, Control, Saline, Immunohistochemistry, Expressing, Comparison